Oligonucleotide probes for specific detection of Giardia lamblia cysts by fluorescent in situ hybridization.

Abstract

Our study focused on the design of oligonucleotide probes and a suitable hybridization protocol that would allow rapid and specific identification of potentially viable cysts of the waterborne parasite Giardia lamblia. Comparative analysis of ribosomal RNA (rRNA) sequences of Giardia lamblia and a number of closely and more distantly related species identified six regions that appear to be specific for the G. lamblia 16S rRNA. Fluorescently labelled probes targeting these regions were produced and employed in fluorescent in situ hybridization (FISH) experiments. Two of the six probes tested successfully. Our study provides the first reported probes for specific FISH detection of G. lamblia. The method depends on sufficient amounts of intact rRNA in the target organism, which is unlikely to be present in nonviable cysts that have been exposed to the environment for a prolonged period. Currently, detection of G. lamblia cysts is largely based on immunofluorescence assays (IFA) targeting cyst wall surface antigens. These assays lack specificity and will detect species others than G. lamblia. Further, IFA will detect nonviable cysts and cyst wall fragments that do not pose a public health risk. In contrast, FISH probes allow specific detection and are likely to only detect viable, infectious cysts.